Ion-exchange and affinity matrices function based on adsorptive purification processes where the matrix selectively binds a target molecule with greater avidity than other molecules present in the same mixture. Such matrices are used to purify and concentrate proteins and other targets from complex, natural, synthetic, and biosynthetic mixtures. These matrices typically consist of polymeric particles (such as cellulose beads) consisting of a packed bed of particles having void spaces through which liquid can flow. A target molecule solution which is to be purified from solution is passed through the packed bed. Binding sites in the particles constituting the packed bed react with materials to be removed from the complex mixture. Upon passing a washing solution through the column, the eluant leaving the column is a purified target solution. The higher the target binding activity, the higher the purification capacity of the packed bed.
Such ion-exchange and affinity matrices may be constructed from a polymeric hydrogel. A polymeric hydrogel consists of an aqueous part (hence the name "hydro-") and a polymer backbone. Generally, a hydrogel has a low-solids content and is very water-like.
Certain features are peculiar to ion-exchange and affinity matrices, respectively, as set forth below.
When a polymeric hydrogel is to be used as an ion-exchange matrix, the matrix is derivatized to make the matrix ionic. The ionic character of the matrix poses a particular problem, as the matrix tends to dissolve over time and become unusable. Generally, a conventional solution to the dissolution problem has been to crosslink the matrix using conventional crosslinking methods which utilize batch chemistry.
Such conventional methods and matrices suffer from certain disadvantages, discussed further below.
The size (diameter) of the cellulose particles used in constructing the hydrogel influences the properties and performance of the hydrogel. Cellulose particles on the order of 450-600 .mu.m are considered large.
Hydrogels for use on a bench scale (i.e., a small scale) have been available. However, there has been an unsatisfied demand for hydrogels that function on to a larger, commercial scale. For use on such a scale, high through-put is important, that is, the highest possible flow rate for each of the steps in column chromatographic processing when operated at the tallest possible column height (i.e., about 1 m versus 0.1 m). These steps comprise loading (adsorption of target molecules to both surface and intraparticle volume), washing of non-target molecules from the media (matrix), elution of target molecules from the matrix, and cleaning (or regeneration) of the matrix.
Designing large scale adsorption media generally calls into play four considerations, namely, (1) void space pressure driven flow; (2) intraparticle transport; (3) site installation; and (4) media stability.
Void space pressure driven flow refers to the pressure needed to sustain a given flow rate in a packed bed chromatographic column.
Intraparticle transport refers to diffusional and/or convective transport of molecules within the hydrogel particle.
Site installation refers to placing certain pre-ordained structures, so that the sites are not too densely packed and are installed at the desired location within the matrix.
Media stability refers to whether the hydrogel dissolves and/or becomes disintiguous over time, so that it has an acceptable shelf-life or easily deforms under ordinary flow rates used in chromatographic processing.
Traditionally, modifying or designing ion-exchange matrices to be useable on a larger scale than bench-scale, such as for commercial production, has posed difficulties that come from the four aspects mentioned above that often are competing. That is, achieving improved performance on one aspect typically disadvantageously has compromised at least one other aspect. Thus, there is a need for a hydrogel for large-scale use which has satisfactory performance optimized in all four aspects.
The stability problem associated with hydrogels has been addressed by chemical crosslinking, to impart chemical and mechanical robustness and to prevent leaching of polymer backbone into the purified product. However, conventional crosslinking procedures improve stability but at the expense of other aspects of the hydrogel.
Particularly, conventional crosslinking methods are known, whereby, using a crosslinking reagent that generally is a bifunctional molecule, a hydrogel that is "outside-in" crosslinked is produced. In conventional chromatographic hydrogels (e.g. Pharmacia Sepharose Fast-Flow ("FF") crosslinked by the conventional "outside-in" crosslinking procedure, extensive crosslinking occurs near the bead surface before crosslinking occurs in the interior of the bead due to installation by batch chemistry.
Conventional crosslinking molecules (e.g., epichlorhydrin) are insoluble in water, which is the solvent used in the conventional "outside-in" procedure. The conventional water-solvent crosslinking process relies on partitioning the crosslinker into the aqueous phase of the hydrogel, and subsequent reaction with the hydrogel polymer backbone. Such phase partitioning is an inefficient mass transfer operation, and results in little penetration of the crosslinking and/or activating molecule into the interior of the bead prior to the reaction of the cross-linker or activating molecule with the matrix.
As a result, "outside-in" crosslinked hydrogels have a higher degree of crosslinking in the outer strata of the particles, and lower crosslinking in the interior of the particles. Excessive crosslinking at the matrix surface can lessen the accessibility to the interior of the bead, i.e., about 70% of the interior volume becomes inaccessible.
Despite the disadvantages of conventional outside-in crosslinking, abandoning crosslinking is not an acceptable solution, because without crosslinking, the matrix becomes not useable because the matrix dissolves or becomes disintiguous over time (because the polymer becomes soluble when stored or operating in aqueous because the matrix is highly ionic) and/or becomes easily deformed when operated in a chromatographic mode. Shelf-life is an important consideration for ion exchange applications of hydrogels. Hydrogels with shelf-lives on the order of many months or years, rather than weeks as conventional hydrogels provide, are desired.
Conventional designs of chromatographic matrices emphasized small particle sizes, so as to reduce intraparticle diffusional mass transfer resistance.
Small particle diameters correspond to higher pressure drops, with the use of low L/D (i.e., length-to-diameter) columns to achieve throughout, which is a disadvantage. For the small particles of the conventional hydrogels, high crosslinking becomes necessary because the pressure necessarily will be so high that otherwise the chromatographic media would be deformed. Overall, considerably less crosslinking is needed for large particles in order to provide resistance to deformation while operated under high flow rates and/or tall columns. There is a need for methods to make larger particles usable in hydrogels, because, generally, larger crosslinked-cellulose particles may have certain practical advantages relative to small particles, such as (1) very low pressure at very high linear velocities; (2) allowing for a process with partially clarified, partially filtered feeds; (3) high throughput at large-scale capacity; and, (4) robustness to sanitization. All of the above should be able to occur in a tall bed height without significant pressure drop.
The sanitization point noted above becomes important because most matrices typically are re-used.
Between purification cycles, matrices typically are cleaned with an NaOH solution with pH of about 12-13 at 45.degree. C. for about 2 to 3 hours, which are relatively harsh conditions. The cleaning (and other chemical treatments) can affect the stability of a matrix, by the reagents disrupting hydrogen bonding.
Also, the need for making larger particles usable in hydrogels further corresponds to the relative advantages of manufacturing large compared to smaller particles, of allowing for (1) continuous processing; (2) simplified classification at high yields; (3) ease of manufacturing; and (4) simple manufacturing for product diversity (e.g., crosslinked DEAE (diethyl amino-ethane) cellulose particles; crosslinked Q cellulose particles; affinity-ligand cellulose particles). However, in the conventional methods, the resulting large particles are outside-in crosslinked and correspondingly suffer from certain disadvantages such as (1) lack of accessibility of submacromolecular species to the interior volume of particles by diffusional and convective transport mechanisms due to molecular exclusion or sieving effects (of these submacromolecular species, i.e., proteins, peptides, etc.); and (2) lack of appropriate site installation into accessible intraparticle domains. Thus, there is a need to overcome the disadvantages associated with large conventionally-crosslinked cellulose particles, without giving up any of the advantages that such conventional particles may provide.
The high degree of crosslinking in the art for all particles, small and large, has particularly made large particles unsuitable for ion-exchange and affinity applications at large-scale (i.e., 1 m tall or higher operated at 1 cm/min or greater linear velocity).
For example, the high degree of crosslinking in the outer strata of the conventional large-bead hydrogels results in minimal intraparticle penetration of average sized protein molecules (such as albumin, 66 kDa) at typical large scale processing linear velocities of 1 cm per minute. Thus, less adsorptive capacity in proteins is seen in large particles crosslinked with classical outside-in methods as applied to small particles (i.e., there is a lack of adsorptive surface area where large particles are used). With large beads, overall less surface area is provided, therefore the need to use the bead interior is increased.
Accordingly, in view of the competing considerations discussed above and not satisfactorily addressed by conventional crosslinking and conventional outside-in crosslinked hydrogels, a crosslinking procedure is needed that gives the stability advantages of conventional outside-in crosslinking methods without at the same time suffering from the disadvantages associated with conventional outside-in crosslinking.
In attempting to scale-up ion-exchange matrices (i.e., to design the matrices for larger scale use), one approach has been to use dimensionless group analysis. Dimensionless group analysis uses the governing physics to generate normalized processing parameters which are dimensionless but scale the relative importance of different phenomena to the process (i.e. the ratio of spatial diffusion to a site to the spatial adsorption of the target molecule once that molecule reaches the site). See R. D. Whitley, K. E. Van Cott, and N. H. L. Wang, Ind. End. Chem. Res. (1993) 32: 149-159. The Whitley paradigm identified the rate limitations in kinetic and mass transfer steps and allowed for rational scale-up of chromatographic processes based on dimensionless ratios of these rates. Whitley et al. (1993) identified the effects of slow sorption kinetics in multi-component systems.
Intraparticle transport and adsorption kinetics are not well characterized or optimized for most commercially available DEAE matrices. Significant intraparticle transport of proteins at processing scale velocities is absent in commercially-available ion exchange matrices. Void-flow convection and/or surface adsorption kinetics has been identified as the rate limiting mass transfer step for many beaded matrices. Thus, the most important dimensionless group for sorptive (i.e., adsorptive) processes is the adsorption number, N.sub.+i, defined by the ratio of sorption kinetic rates to the convection rate. That is, ##EQU1## where L is the column length; C.sub.i is the concentration of species i; k.sub.+i is the adsorption rate coefficient of i; and u.sub.o is the average linear velocity of the fluid in the void space.
Considering the processing variables which affect the N+i expression, it can be seen that column length (L) and linear velocity (u.sub.o, which normalizes volumetric processing rates to the column length to bead and column contacting times) are important in scaling up a chromatographic process from the lab bench to production scale.
Benefits of long column lengths have been recognized. Particularly, with long column length, constant pattern behavior (i.e., steady state plug flow) can be approached. Under conditions of constant pattern the highest possible concentration driving force for adsorptive or desorptive processes occurs. In practical terms, that translates into efficient adsorption and higher capacity; sharper concentration fronts; increased eluted product concentration; less elution and wash volumes; and increased productivity when operated in a long column.
Numerical simulations have shown that for N.sub.+i .gtoreq.10, the system can be considered nearly at equilibrium, according to Whitley et al. Under these conditions, the sorption kinetics are not rate limiting and there will be little product loss due to inefficient adsorption or peak spreading. Thus, efficient chromatographic processes should have high N.sub.+i numbers (high ratios of L to u.sub.o) for the adsorption step.
It has been shown experimentally that the N.sub.+i variable is the key design variable.
Previously, the present inventors have found that a certain phenomenon governs processing goals and that to optimize scale-up, resolution, and product yields, as well as to develop novel matrices for the isolation of "troublesome proteins", chromatographic processes may be tailored to take advantage of "N". Particularly,
N.sub.+i .gtoreq.10 for column loading step PA1 N.sub.-i &lt;&lt;10 for column washing step PA1 N.sub.-i .gtoreq.10 for column elusion step
In the above, i represents any given species (molecule) to be adsorbed, with "+" meaning "adsorption", and "-" meaning "desorption".
Putting the adsorption number theory to work has posed difficulties, because hydrogel matrices for anion exchange adsorption chromatography of proteins frequently incorporate small particles (&lt;100 .mu.m mean particle size) that have a short path length for diffusional transport of target proteins. As a result, high pressure drops, low flow rates, and low L/D column dimensions usually accompany this design emphasis. Thus, there is little room to manipulate N.sub..+-.i when doing process scale-up (i.e. increasing column length at constant processing velocity so as to increase N.sub..+-.i). Large diameter particles (.about.600 .mu.m) engineered to have minimal intraparticle transport limitations provide the flexibility of high L to u.sub.o and thus maximal N.sub.+i.
A model for optimizing affinity media was demonstrated using large diameter (500-700 .mu.m) cellulose particles with relatively low solids contents (Kaster et al., 1994). That optimization yielded an adsorptive media which provided: (i) low pressure drops at high flow rates in a high L/D column mode operation; and, (ii) rapid transport to adsorption sites.
A high L/D column, coupled with the wide range of flow rates available due to minimal pressure drop, allow the user to manipulate the N.sub.+i number to a greater extent than for commercially available matrices, would allow for the design of more efficient and productive chromatographic processes. See Whitley et al. (1993); see also J. A. Kaster, W. Oliveira, W. G. Glasser and W. H. Velander, Optimization of pressure-flow limits, strength, intra-particle transport and dynamic capacity by hydrogel solids content and beads size in cellulose immunosorbents, J. Chromatography 79-90 (1993).
Thus, a need remains for further methods to optimize hydrogel matrices for large scale protein purification.
Also, conventional matrices would benefit from methods for enhancing ion exchange performance, including methods for exploiting relative rates of mass transfer and sorption kinetics (sorption number N.sub..+-.i).
Particularly, there is a need for a method of improving the shelf life and deformability of large crosslinked cellulose particles to be used in ion-exchange matrices, especially to bring the shelf-life to the order of many months or years rather than weeks as is the shelf-life for conventionally crosslinked or uncrosslinked ion exchange cellulose particles.
In the case of affinity applications for cellulose particles, there is a need to improve ligand spacing in the cellulose particles.
In affinity applications, getting to the core of the bead is even more important than in the case of ion-exchange matrices, because of the relative number of sites. Sites must be functional. If sites are installed too close together, they will be dysfunctional.
Thus, to summarize the above, there have been many needs for an improved hydrogel and for improved methods of producing hydrogels, particularly for installation of crosslinking or activation chemistries (used to attach affinity ligands).
Additionally, at the same time, in the context of products containing large macromolecular complexes (i.e. particles larger than about 10 nm hydrodynamic radius) such as viruses and other pathogens (e.g., large viral particles such as HIV, Hepatitis B and C) there has been a need for improved methods for selectively removing such pathogens from feedstreams. Existing methods have suffered from various shortcomings, such as loss of valuable feedstream components having hydrodynamic radii of about 5 nm or less (therapeutic proteins).